Immune regulation is a key component of an efficient adaptive immune system both to avoid pathology to self-antigens and collateral damage during infectious challenges. CD4+ CD25+ regulatory T cells (Tregs) are emerging as key regulators of immune responses. Although first described to regulate peripheral self-tolerance and to protect against autoimmunity, the CD25+ Tregs have also been implicated in suppressing immune responses to infections, tumors and transplants. Little is known of the mechanism whereby naturally occurring CD4+CD25+ regulatory T cells downregulate immune function. This proposal seeks to define the mechanism whereby the target T cell activation program is terminated by Tregs and the functional consequences of this encounter. We have made three key observations that give us a novel handle on the suppression process. First, Tregs were suppressive within a surprisingly narrow kinetic window: necessary and sufficient in the first 6-1 Oh of culture, prior to cell division. Importantly, the timing of suppression was dictated by the kinetics and quality of target T cell activation, rather than Treg stimulation. Indeed, suppression occurred rapidly (1-2 h) if Tregs were added to existing 6h activated target T cell cultures. The tight kinetics provides a unique opportunity to define the molecular events that lead to target T cell shutdown. Second, Tregs induced a distinct transcriptional program in target T cells that differs from simple IL-2 deprivation, TGF[unreadable] treatment, anergy or death mechanisms but that is exemplified by genes associated with proliferative arrest. Third, with provision of co-stimulation, target T cells escaped proliferative arrest but still succumbed to a Treg block in Th differentiation suggesting that the two forms of Treg-suppression are distinct molecular events. We will test the following hypothesizes: Specific Aim 1. Tregs terminate an existing target T cell activation program. Specific Aim 2. Tregs modulate specific signal pathways in part through enhancement of proteolytic degradation of signaling proteins. Specific Aim 3. Tregs induce a distinct group of negative regulators of IL-2 and/or proliferation. Specific Aim 4. Treg-suppression of target T cell proliferation and differentiation are independently regulated, distinct molecular events. Relevance to human health: Regulatory T cells are important regulators of immune responses but how they function is unknown. A better understanding of how they disable the immune system will allow the design of therapeutics that render targets more susceptible to suppression in patients with autoimmunity and resistant to suppression to help immune destruction of tumors.